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1. EP3487540 - SITE-SPECIFIC CONJUGATION TO ANTIBODY LYSINE RESIDUES WITH SOLID-PHASE IMMOBILIZED MICROBIAL TRANSGLUTAMINASE MTG AND MTG IN SOLUTION

Office European Patent Office
Application Number 17742684
Application Date 11.07.2017
Publication Number 3487540
Publication Date 29.05.2019
Publication Kind A1
IPC
A61K 47/64
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
47Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
50the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
51the non-active ingredient being a modifying agent
62the modifying agent being a protein, peptide or polyamino acid
64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
A61K 47/68
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
47Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
50the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
51the non-active ingredient being a modifying agent
68the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
C12N 11/08
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
11Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
02Enzymes or microbial cells immobilised on or in an organic carrier
08the carrier being a synthetic polymer
CPC
C12N 11/08
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
11Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
02Enzymes or microbial cells immobilised on or in an organic carrier
08the carrier being a synthetic polymer
A61K 47/64
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
47Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
50the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
51the non-active ingredient being a modifying agent
62the modifying agent being a protein, peptide or polyamino acid
64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
A61K 47/6811
AHUMAN NECESSITIES
61MEDICAL OR VETERINARY SCIENCE; HYGIENE
KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
47Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
50the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
51the non-active ingredient being a modifying agent
68the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
6801Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
6803Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
6811the drug being a protein or peptide, e.g. transferrin or bleomycin
Applicants SCHERRER INST PAUL
Inventors SPYCHER PHILIPP RENÉ
BEHE MARTIN
SCHIBLI ROGER
HURWITZ DAVID
KREIS OLIVIER
Designated States
Priority Data 16180382 20.07.2016 EP
Title
(DE) STELLENSPEZIFISCHE KONJUGATION AN ANTIKÖRPER-LYSIN-RÜCKSTÄNDE MIT FESTPHASENIMMOBILISIERTER MIKROBIELLER TRANSGLUTAMINASE-MTG UND MTG IN LÖSUNG
(EN) SITE-SPECIFIC CONJUGATION TO ANTIBODY LYSINE RESIDUES WITH SOLID-PHASE IMMOBILIZED MICROBIAL TRANSGLUTAMINASE MTG AND MTG IN SOLUTION
(FR) CONJUGAISON SITE-SPÉCIFIQUE SUR DES RÉSIDUS LYSINE D'ANTICORPS À L'AIDE D'UNE TRANSGLUTAMINASE MICROBIENNE MTG IMMOBILISÉE EN PHASE SOLIDE, ET D'UNE MTG EN SOLUTION
Abstract
(EN)
Site-specific modification of proteins with microbial transglutaminase (MTG) is a powerful and versatile strategy for a controlled modification of proteins under physiological conditions. We present evidence that solid-phase microbead- immobilization can be used to site-specifically and efficiently attach different functional molecules important for further downstream applications to proteins of therapeutic relevance including scFV, Fab-fragment and antibodies. We demonstrate that MTG remained firmly immobilized with no detectable column bleeding and that enzyme activity was sustained during continuous operation, which allowed for a convenient recycling of the enzyme, thus going beyond solution-phase MTG conjugation. In addition it is showed that immobilized MTG shows enhanced selectivity towards a certain residue in the presence of several reactive residues which are all targeted if the conjugation was carried out in solution. It is also reported on the site-specific lysine conjugation of antibodies using potent glutamine containing peptides with immobilized and MTG in solution. In addition, the generation of dual site-specifically conjugated IgG1 with immobilized and MTG in solution is reported, i.e. site-specific conjugation to glutamine and lysine residues of IgG1 antibody. Site-specific glutamine conjugation with small peptides containing a lysine residue and a functional moiety is also described.

(FR)
La modification site-spécifique de protéines à l'aide d'une transglutaminase microbienne (MTG) est une stratégie puissante et polyvalente pour la modification contrôlée de protéines dans des conditions physiologiques. Nous avons apporté des preuves montrant que l'immobilisation de microbilles en phase solide peut être utilisée pour lier efficacement et de façon spécifique au site diverses molécules fonctionnelles, importantes pour des applications supplémentaires en aval, à des protéines présentant une pertinence thérapeutique, lesquelles comprennent le scFV, les fragments Fab et les anticorps. Nous avons démontré que la MTG restait fermement immobilisée sans perte détectable au niveau de la colonne et que l'activité enzymatique était maintenue lors d'un fonctionnement en continu, ce qui permet un recyclage pratique de l'enzyme, allant ainsi au-delà de la conjugaison de la MTG en phase solution. En outre, il a été montré que la MTG immobilisée présentait une sélectivité améliorée envers un résidu donné en présence de plusieurs résidus réactifs, lesquels sont tous ciblés si la conjugaison est réalisée en solution. L'invention décrit également la conjugaison spécifique au site d'anticorps au niveau de résidus lysine par l'utilisation de peptides puissants contenant de la glutamine avec une MTG immobilisée et une MTG en solution. De plus, l'invention décrit la génération d'IgG1 doublement conjugué de façon site-spécifique par une MTG immobilisée et en solution, ceci correspondant à la conjugaison spécifique au site de résidus glutamine et lysine de l'anticorps IgG1. L'invention concerne également la conjugaison spécifique au site au niveau de la glutamine avec de petits peptides contenant un résidu lysine et une partie fonctionnelle.